Celiac disease is an immune system disorder, genetic in origin, induced by gluten consumption, which is a protein found present in wheat, rye and barley. This is poorly digested in the human upper gastrointestinal tract. Gluten is composed of two fractions, gliadin and glutenin. Gliadins are alcohol soluble and contain the largest amount of toxic components for celiac patients (Green, P. H. R. and Cellier, C. 2007. Celiac disease. N. Engl. J. Med.; 357:1731-1743).
When a patient with celiac disease eats some gluten-containing food, their immune system responds in such a way that damages or destroys the intestinal villi, causing the food nutrients to be non-absorbed, thereby leading to a poor nutrition. The symptoms of celiac disease vary depending on the age of the patient, in children being the most common diarrhea, abdominal distension, vomiting and weight loss, while in adults the prevailing symptoms are iron-deficiency anemia, fatigue, bone pain, arthritis, osteoporosis, among others (National Digestive Diseases Information Clearinghouse, NIH Publication No. 08-4269, 2008).
Until recently, celiac disease was considered a rare disease, but currently it is one of the most common intolerances, having a global incidence of 1 in every 150 newborns, and it is estimated that only 9% is diagnosed. According to numbers provided by the National Institute of Medical Sciences and Nutrition (Instituto Nacional de Ciencias Médicas y Nutrición) Salvador Zubirán, in Mexico, there is an approximate volume of 2.6 million potential celiac patients (http://celiacosdemexico.org.mx/manifiesto-celiaco).
Currently, the only accepted treatment for celiac disease is a gluten-free diet for life. According to the Codex Alimentarius, it is considered that a food is gluten-free when the gluten concentration therein is less than 20 ppm. However, this represents negative nutritional implications such as a decreased intake of polysaccharides and thus lower energy intake, a reduction in beneficial intestinal flora for human health and an increase in the presence of opportunistic pathogens. Beneficial flora reduction by the gluten-free diet negatively affects immunostimulating activity and decreases the production of anti-inflammatory compounds.
Therefore, there is a need to have gluten-free products, suitable for consumption by celiac patients and, accordingly, allowing them to improve their diet by having a greater variety of foodstuffs at hand.
One of the most important products in the daily diet of human beings is bread, manufactured mainly from wheat flour.
The properties of the wheat dough depend primarily on gluten proteins. In recent years, diverse treatments have been applied to improve the quality of these proteins or to improve the gas retention process to produce a more aerated bread (Arendt et al., 2007. Impact of sourdough on the texture of bread. Food Microbiology 24:165-174). One of these processes is the production of bread using sourdoughs.
The main function of this process is to leaven the dough to produce a higher gas content and therefore a fluffier bread having softer crumbs.
Sourdough is obtained with a fermented mixture of flour (wheat, oat and rice, etc.) and water with yeast and lactic acid bacteria (LAB) usually belonging to the genus Lactobacillus (De Vuyst, L. and Vancanneyt, M. 2007. Biodiversity and identification of sourdough lactic acid bacteria. Food Microbiology 24.120-127). The use of sourdoughs offers great advantages in good baking technology, for example, pH decrease during fermentation, better gas retention, higher resistance of the gluten network, inhibition of fluor amylases, binding of water with gluten and starch granules, swelling of pentoses, solubilization of phytate complex and prevention of a poor fermentation (Di Cagno, R. et al. 2002. Proteolysis by sourdough lactic acid bacteria: Effects on wheat Flour protein fractions and gliadin peptides involved in human cereal intolerante. Applied and Environmental Microbiology 68: 623-633). These sourdoughs are admixed with fresh dough for bread making.
In addition, the LAB used in fermentation with sourdoughs allows gluten modification during bread elaboration, thereby allowing the removal of protein fractions that are toxic for celiac patients. However, not all the lactic acid bacteria can decrease the gluten residual concentration to doses capable of being tolerated by gluten-intolerant people (celiac patients), whereby it is necessary to select the LAB type and to find a suitable combination there between, by using proteases co-adjuvating in gluten hydrolysis.
During fermentation, the LAB proteolytic system releases low molecular weight peptides and amino acids, promoting the metabolic activity of microorganisms, contributing to achieving a better flavor and reducing the content of allergenic peptides (De Angelis, M. et al. 2005. VSL #3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsable for Celiac Sprue probiotics and gluten intolerante. Biochimica et Biophysica Acta 1762(1): 80-93), which opens a great hope for celiac patients sensitive to the gliadin fraction, reason why they have been prevented from ingesting products containing even small amounts of gluten (Wieser, H. 2007. Chemistry of Gluten proteins. Food Microbiology 24: 115-119; Di Cagno et al., 2002). Studies carried out in bread making from sourdough showed that the LAB, under specific processing conditions (long time fermentation and semi-liquid), have the ability to hydrolyse the wheat gliadin fraction (Di Cagno, R. et al. 2004. Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients. Applied and Environmental Microbiology 70(2):1088-1096.; De Angelis et al., 2005).
Rizzello et al., (Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease. Applied and Environmental Microbiology 73 (14): 4499-4507, 2007) used mixtures of non-commercial strains of Lactobacilli (previously selected based on their ability to hydrolyze gliadins) with fungal origin proteases in different combinations, in sourdoughs to remove wheat flour toxicity during a relatively long fermentation. It was noted that hydrolysis kinetics by Lactobacilli was highly efficient, further, the proteins extracted from the sourdough induced interferon γ activation; the albumins, globulins and gliadins were completely hydrolyzed, while 20% of the glutenins remained in the sourdough.
Likewise, US Patent Application No. US 2008/0131556 describes a mixture of at least six commercially available LAB and/or bifidobacteria species. This mixture can be used in the preparation of sourdoughs. Similarly, when a sufficient amount of microbial proteases commonly used in bakery is added to these formulations, the fermented sourdough has a gluten concentration lower than 200 ppm, which might be non-appropriate for the consumption by a celiac patient. Likewise, the mixture to achieve this degradation is complex because it is necessary to use a large number of species, since the more LAB species are used, the higher degradation of gliadins is seen.
On the other hand, International Publication No. WO 2010/073283 describes a mixture comprising two types of LAB, in combination with one or more fungal proteases. After a 12 h fermentation, no traces of gliadin and glutenin were detected. In addition, the gluten residual concentration was less than 20 ppm.
As can be seen, bacterial cultures already known in the prior art for gluten degradation present certain disadvantages, such as the use of LAB complex mixtures (of at least six species) in the case of commercially available strains, or the use of specifically selected strains, involving the need to activate, reproduce, wash and add the culture in suspension (inoculum) to the dough, requiring daily preparation thereof and a highly specialized management to prevent contamination, keeping it active in the same growing stage and in the proper ratio between LAB species and in a sufficient amount, which represents a latent risk of contamination.
An alternative to reduce the difficulties related to the inoculum elaboration each time this is required is to microencapsulate it in order to produce a sufficient amount, involving a reduction in culture handling, thereby reducing risks related to contamination and viability of the lactic-acid cultures and promoting process efficiency for the degradation of wheat doughs.
The LAB viability and activity is determined by a series of factors, including the rate of culture multiplication, lactic and acetic acid production capacity, total solid content, temperature and time of incubation, amount of inoculum used, and antibiotic, disinfectant or detergent residues (Picot and Lacroix, 2003; Briceño 2005; Desmond et al., 2002; Favaro-Trinidad and Grosso, 2002; Lian et al., 2003; Akalin et al., 2004; Cerdeira et al., 2005; Iyer and Kailasapathy, 2005; Ozer et al., 2005; Ann et al., 2007).
There are different methods to increase prebiotic bacteria resistance to adverse conditions, for example, microencapsulation and incorporation of micro-nutrients with prebiotic function (Picot and Lacroix, 2003; Chandramouli et al., 2004; Talwalkar and Kailasapathy, 2004; Cerdeira et al., 2005; Iyer and Kailasapathy, 2005; Ann et al., 2007).
Microencapsulation has been proposed to increase prebiotics viability, since their sensitivity to the oxygen high levels, manufacturing, storage, freezing, acidic or alkaline conditions during the passing through the gastrointestinal tract may be protected (Favaro-Trinidad and Grosso, 2002; Picot and Lacroix, 2003; Chandramouli et al., 2004; Iyer and Kailasapathy, 2005; Crittenden et al., 2006; Ann et al., 2007).
Even when the microencapsulation of LAB is already known in the state of the art, in order to ensure good protection for the materials to be encapsulated, which will be used during sourdough fermentation to degrade the gluten, it is essential to make a good selection of the drying conditions as well as of the encapsulating media, which is not described in the state of the art, since the micro-capsule wall design is a critical point performed depending on the specific type of material to be protected, the environment the micro-capsules should be applied on, and the release mechanism of the active material they protect.